This project focuses upon the molecular genetics of two systems of protein - DNA interaction: First, the recognition by RNA polymerase of sequences in DNA encoding transcription initiation sites (promoters); second, the recognition by Trp holorepressor of its cognate operator. A family of mutationally connected constitutive promoters lying within the trpD gene of Escherichia coli will be analyzed by the techniques of molecular cloning, restriction endonuclease cleavage, and DNA sequence determination. The effects of specific single base pair changes on the parameters of interaction with RNA polymerase will be established. The techniques of molecular cloning have enabled us to isolate, for the first time, the structural gene for a biosynthetic repressor protein, namely TrpR. This gene is contained within a 1250 base pair fragment cloned into the amplifiable plasmid pBR322. By the techniques of DNA sequence determination, we will establish the primary structure of Trp repressor protein. Plasmid derivatives capable of mediating high-level production of Trp repressor protein will be constructed. Pure preparations of Trp repressor protein will be prepared, then used to probe the mode of association of this protein with trp operator and L-tryptophan, the effector molecule.